Programmed Death 1 (PD-1), a member of the CD28 costimulatory gene family, is moderately expressed on naive T, B and NKT cells and up-regulated by TB cell receptor signaling on lymphocytes, monocytes and myeloid cells (1). PD-1 has two known ligands with distinct expression profiles, PD-L1 (B7-H1) and PD-L2 (B7-DC). PD-L2 expression is relatively restricted and is found on activated dendritic cells, macrophages and monocytes and on vascular endothelial cells (1-3). In contrast, PD-L1 is expressed more broadly including on naive lymphocytes and its expression is induced on activated B and T cells, monocytes and dendritic cells. Furthermore, by mRNA, it is expressed by non-lymphoid tissues including vascular endothelial cells, epithelial cells and muscle cells.
PD-1 is recognized as an important player in immune regulation and the maintenance of peripheral tolerance. In the mouse, this was shown to require PD-L1 expression on peripheral tissues and ligation of PD-1 on potentially autoreactive T cells to negatively modulate T cell activation involving an ITIM sequence in the PD-1 cytoplasmic domain (1, 4).
Depending on the specific genetic background, pdcd1−/− mice spontaneously develop lupus-like phenomena or dilated cadiomyopathy (5, 6). Furthermore, antibody-induced blockade of the PD-1/PD-L 1 pathway was demonstrated to accelerate the onset of autoimmune insulitis and diabetes in NOD mice (7).
Human cancers arising in various tissues were found to over-express PD-L1 or PD-L2. In large sample sets of e.g. ovarian, renal, colorectal, pancreatic, liver cancers and melanoma it was shown that PD-L1 expression correlated with poor prognosis and reduced overall survival irrespective of subsequent treatment (15-26). Similarly, PD-1 expression on tumor infiltrating lymphocytes was found to mark dysfunctional T cells in breast cancer and melanoma (27-28) and to correlate with poor prognosis in renal cancer (29). Using primary patient samples, it was shown that blockade of PD-1 or PD-L1 in vitro results in enhancement of human tumor-specific T cell activation and cytokine production (30). Consequently, in several murine syngeneic tumor models, blockade of either PD-1 or PD-L1 significantly inhibited tumor growth or induced complete regression.
A PD-1 blocking mAb (h409A11) was discovered and developed for use to treat human cancer patients and chronic virus-infected patients (described in co-pending application WO2008/156712).
Antigen-specific T cell dysfunction or tolerance is exemplified by the accumulated loss of the potential to produce Interleukin 2 (IL-2), Tumor Necrosis factor (TNF) α, perforin, interferon (IFN) γ (8) and inability to mount a proliferative response to T cell receptor triggering (1). The PD-1 pathway controls antigen-specific T cell tolerance and was found to be exploited in viral infection and tumor development to control and evade effective T cell immunity.
In chronic infection with LCMV (mouse), HIV, HBV or HCV (human), antigen-specific T cells were found to express aberrantly high levels of PD-1 correlating with their state of anergy or dysfunction (9). Blocking the PD-1-PD-L1 interaction in vivo (LCMV) or in vitro (HIV, HCV, HBV) was shown to revive anti-viral T cell activity (10-12). PD-1 blockade in recently Simian Immunodeficiency Virus-infected macaques resulted in strong reduction of viral load and increased survival (13). Similarly, reduction in viral load was confirmed in second study using long-term SIV-infected rhesus macaques (14).
Overall, the PD-1/PD-L1 pathway is a well-validated target for the development of antibody therapeutics for cancer treatment. Anti-PD-1 antibodies are also useful for treating chronic viral infection. Memory CD8+ T cells generated after an acute viral infection are highly functional and constitute an important component of protective immunity. In contrast, chronic infections are often characterized by varying degrees of functional impairment (exhaustion) of virus-specific T-cell responses, and this defect is a principal reason for the inability of the host to eliminate the persisting pathogen. Although functional effector T cells are initially generated during the early stages of infection, they gradually lose function during the course of a chronic infection. Barber et al. (Barber et al., Nature 439: 682-687 (2006)) showed that mice infected with a laboratory strain of LCMV developed chronic infection resulting in high levels of virus in the blood and other tissues. These mice initially developed a robust T cell response, but eventually succumbed to the infection upon T cell exhaustion. The authors found that the decline in number and function of the effector T cells in chronically infected mice could be reversed by injecting an antibody that blocked the interaction between PD-1 and PD-L1.
PD-1 has also been shown to be highly expressed on T cells from HIV infected individuals and that receptor expression correlates with impaired T cell function and disease progression (Day et al., Nature 443:350-4 (2006); Trautmann L. et al., Nat. Med. 12: 1198-202 (2006)). In both studies, blockade of the PD-1 pathway using antibodies against the ligand PD-L1 significantly increased the expansion of HIV-specific, IFN-gamma producing cells in vitro.
Other studies also implicate the importance of the PD-1 pathway in controlling viral infection. PD-1 knockout mice exhibit better control of adenovirus infection than wild-type mice (Iwai et al., Exp. Med. 198:39-50 (2003)). Also, adoptive transfer of HBV-specific T cells into HBV transgenic animals initiated hepatitis (Isogawa M. et al., Immunity 23:53-63 (2005)). The disease state of these animals oscillates as a consequence of antigen recognition in the liver and PD-1 upregulation by liver cells.
Therapeutic antibodies may be used to block cytokine activity. A significant limitation in using antibodies as a therapeutic agent in vivo is the immunogenicity of the antibodies. As most monoclonal antibodies are derived from non-human species, repeated use in humans results in the generation of an immune response against the therapeutic antibody. Such an immune response results in a loss of therapeutic efficacy at a minimum, and potentially a fatal anaphylactic response. Accordingly, antibodies of reduced immunogenicity in humans, such as humanized or fully human antibodies, are preferred for treatment of human subjects. Exemplary therapeutic antibodies specific for human PD-1 are disclosed in commonly-assigned U.S. Patent Application Publication No. US2010/0266617, and in International Patent Publication No. WO2008/156712, the disclosures of which are hereby incorporated by reference in their entireties.
Antibodies for use in human subjects must be stored prior to use and transported to the point of administration. Reproducibly attaining a desired level of antibody drug in a subject requires that the drug be stored in a formulation that maintains the bioactivity of the drug. The need exists for stable formulations of anti-human PD-1 antibodies for pharmaceutical use, e.g., for treating various cancers and infectious diseases. Preferably, such formulations will exhibit a long shelf-life, be stable when stored and transported, and will be amenable to administration at high concentrations, e.g. for use in subcutaneous administration, as well as low concentrations, e.g. for intravenous administration.